The routine production of monoclonal antibodies relies on the fusion, or hybridization, of tumor cells, such as myeloma cells or virus-transformed cells and antibody secreting cells, such as those found in spleen or peripheral blood. The resulting hybridoma produces the specific antibodies of the blood or spleen cell parent while retaining the longevity in culture, and tumor-forming ability in animals, of the tumor cell parent. The procedure suffers from a number of limitations primarily arising from the fact that the tumor cell line must be a mutant line having certain characteristics which are only rarely found.
One requirement is that the tumor cells do not secrete infectious virus, so that the antibodies can be used with safety in treating patients. It would also be desirable, although with modern separation methods not critical (as will be exemplified), to select a tumor line that does not secrete immunoglobulins.
Another limitation arises from the requirement with present procedures that there be a method for selecting the hybridoma cells away from unfused tumor cells. As reported by M. D. Scharff et al. in "Hybridomas as a Source of Antibodies", Hospital Practice, Jan. b 1981, 61-11, only about one out of every 200,000 spleen cells actually forms a viable hybrid with a myeloma cell. Accordingly, the unfused cell and the myelomamyeloma hybrids must be selectively eliminated (there is no need to eliminate unfused spleen cells because they die out in culture and are rapidly overgrown by the hybrids), which is usually accomplished by culturing the tissue cells in HAT (hypoxanthine, aminopterin and thymidine) medium. The myeloma or tumor cells are selected so as to lack HGPRT (hypoxanthine guanine phosphoribosyl transferase), and/or thymidine kinase (TK), enzymes the cell requires in order to use exogenous hypoxanthine and thymidine to synthesize purines. Cells lacking HGPRT and/or TK die when grown in the presence of aminopterin, which blocks endogenous synthesis of purines. Myeloma or tumor cells that have been fused to spleen or peripheral blood cells containing HGPRT and TK are able to use the hypoxanthine and thymidine in the HAT medium and therefore survive. Accordingly, the HAT solution is utilized as a means to select the fused cells from the background of unfused and nonhybridoma cells. In order to obtain a tumor cell line that is deficient in HGPRT and/or TK, one must select the tumor cells for azaguanine, thioguanine or bromodeoxyuridine resistance. Those cells containing HGPRT and TK enzymes take up the toxic nucleotide analogs during culturing and die, while the resistant cells survive. Thus, the tumor line must be one which not only does not secrete infectious virus, and preferably one which does not secrete immunoglobulins, but it must also be azaguanine, thioguanine or bromodeoxyuridine resistant (i.e., selectable in HAT medium).
There are significant reasons for requiring HAT selectability related to the fact that fusion rates are generally low, as indicated by Scharff et al. supra; because of a limited number of successful fusions, the culture is quickly overrun by unfused tumor cells. The low rate of fusion results at least in part from the fact that the standard fusion agent, polyethylene glycol (PEG), is in fact relatively toxic to cells. Methods not involving the use of an enzyme deficient tumor line are known. Recently, there has been reported the use of diethylpyrocarbonate to pretreat tumor cells. Unfused tumor cells treated with diethylpyrocarbonate will die. However, diethylpyrocarbonate itself is quite toxic to the fused cells, resulting in a very low rate of fusion, "Production of Human B-Cell Hybridomas from Patients with SLE", Littman et al., Arth. Rheum. 25; No 4 (Suppl.): 528 (1982). Other researchers have successfully used lipid vesicles for intratype fusion. See, in this regard, D. Papahadjopoulas et al., "Fusion of Mammalian Cells by Unilamelar Lipid Vesicles: Influence of Lipid Surface Charge, Fluidity and Cholesterol", Biochimica et Biophysica Acta, 323:23-42 (1973); R. C. McDonald et al., "Interactions Between Lipid Vesicles and Cell Membranes", Ann. N.Y. Acad. Sci., 308: 200-214 (1978); and F. J. Martin et al., "Lipid Vesicle-cell Interactions--II. Induction of Cell Fusion", The Journal of Cell Biology, 70:506-514 (1976). Such researchers have concluded that successful lipid bridged fusion in vitro depends strongly upon calcium ion-induced phase changes. (See F. J. Martin et al., supra, and D. Papahadjopoulos et al., "Calcium-Induced Lipid Phase Transitions and Membrane Fusion", Ann. N.Y. Acad. Sci., 308:50-66 (1978).)
On the other hand, G. Poste et al., "Membrane Fusion", Biochimica et Biochysica Acta, 300:421-45 (1973), attempt to describe what they perceived as a natural mechanism for cell fusion during a large number of cellular and subcellular activities. In referring to natural intratype in vivo cell fusions, key importance is attributed to the displacement of calcium ions from the cell membranes. The authors also indicate that cholesterol plays an adverse role in restricting phospholipids, thus inhibiting cell fusion.
In general, the fusion agent of choice in the creating of hybridomas has been PEG, and in all standard lymphocyte fusion procedures various concentrations and/or molecular weights of PEG are used as the fusing agent. During the first hours and days after fusion, the critical period when hybridized cells are in danger of being overgrown by unfused tumor cells, the hybrids are at a distinct disadvantage. It is for this reason that a means for selecting against the tumor cells has been required. If it were possible to avoid the toxic effect of PEG and still achieve fusion, "selectable" tumor lines would not necessarily be needed.
Another reason for low fusion rates lies in the fact that the mutant tumor lines, selected for HAT sensitivity, very often have poor inherent fusion rates. Despite several years of work by a number of investigators, only several selectable fusion lines are available, none of which actually works very well. On the other hand, if the line did not have to be HAT selectable, a large and variable group of useful tumor lines would be immediately available for use.
The present application is directed to a procedure which does not rely upon a HAT selectable tumor line by avoiding the use of PEG in the fusion process. Rather, a more physiological approach has been taken involving a plurality of steps that results in destabilization of the target cell membranes, the formation of bridges between the cells, and cell fusion. Even though Poste et al., supra, have described the natural in vivo mechanism of cell fusion as including displacement of calcium ions from the membranes, when prior workers, such as Papahadjopoulas et al., supra, have attempted to fuse cells of the same type in vitro using phospholipid vesicles, they have had to increase the presence of calcium ions; depletion has been contraindicated. In contrast, we have discovered that in order to achieve intertype fusion in vitro using a fusogen such as a phospholipid, one must substantially remove at least calcium ions, to destabilize the cell membranes; thereafter polyvalent ions, such as calcium ions, are added back.
The procedure of the present invention is efficient and quick, resulting in viable and rapidly dividing hybridized cells after as short a period as 24 hours. When using a nonselectable tumor line as the fusion partner, no overgrowth of hybrids is found due to the extremely rapid growth of the hybrid cells. Accordingly, the hybridized cells can readily be separated from tumor cells present in the same cultures by limiting dilution cloning, i.e., cultures are diluted to a concentration allowing theoretical distribution of one cell per tissue culture well. All cells subsequently arising in that well have the same parent and are genetically identical.
In one embodiment, a chelating agent is added to a mixture of tumor cells and cells which secrete a desired endogenous protein or other desired biologically active substance, to substantially remove at least calcium ions, preferably ions of both calcium and magnesium, from the cell environment, whereby to destabilize the membranes of the cells. Alternatively, calcium and magnesium ions may be substantially removed by repeatedly washing the cells in the fusion mixture with calcium and magesium-free medium, i.e., by repeated centrifugations and resuspensions in the described medium.
In another embodiment, microcells containing one to ten chromosomes are produced from cells having the capacity to produce the desired biologically active substance and mixed with the tumor cells. In this embodiment, removal of calcium ions or calcium and magnesium ions destabilizes the tumor cells, following which microcells are treated to remove divalent cations. The microcells and tumor cells must be treated separately because of the need for different modes of centrifugation occasioned by the difference in size between microcells and tumor cells. The microcells are produced by a process which includes the steps of blocking the growth of the cells in the metaphase stage, disrupting the nuclear membrane and reforming a membrane around one to ten chromosomes.
A fusogen, for example, a phosphoglyceride, is added to the destabilized cells or mixture of destabilized tumor cells and microcells and then polyvalent cations, including calcium ions, are added back to the mixture of fusogen and destabilized cells or fusogen, destabilized tumor cells and microcells, whereby to obtain fused cells, such as hybridomas or microcell transerted tumor cells.
It will be appreciated that the fusion procedure of this invention has broad application and can be used to fuse cells that produce metabolites other than antibodies. In broader concept, many other cell types and many forms of microcells can be "immortalized" by fusion with appropriate tumors, making the commercial preparation of such proteins as interferons, hormones, and the like vastly more efficient. By using the present procedure, hybridoma technology can be used to produce materials that are otherwise producible only with the techniques of recombinant DNA. In its broader form, with the cells secreting their own natural product, albeit in an indefinite unregulated manner, the process of reaching commercial production levels would be quicker and easier. Candidates for fusion are readily obtainable since time and effort would not be required to search for suitable "selectable" tumor mutants, but presently available tumor lines can be immediately used.
In accordance with this broader statement of the present invention, we provide a method for preparing fused cells that produce, for example, endogenous proteins such as antibodies, interferons, lymphokines, hormone precursors, hormones or other biologically active substances. Insulin, growth hormone, L-thyroxin, estradiol, testosterone, hydroxycortisone and cortisol are only a few examples of such substances. The process entails mixing together (a) first, tumor cells that do not produce, for example, endogenous proteins of the selected type; and (b) second, mammalian cells that are capable of producing the selected protein or microcells produced from such cells. Usually the second cells are non-tumor mammalian cells but a tumor-tumor hybridoma will also be illustrated.
Calcium and magnesium ions are substantially removed from the cell environment, either by chelation or by repeated washing, whereby to destabilize the membranes of the cells. A fusogen is added to the destabilized cells and then polyvalent cations are added back to the mixture of fusogen and destabilized cells, whereby to obtain by fusion hybridomas of the tumor cells and non-tumor cells or microcell transerted tumor cells. Finally the hybridomas or microcell transerted tumor cells are reproduced by standard cloning procedures.